The invention relates to a method for the resolution of N-5-formyl-(6R,S)-5,6,7,8-tetrahydrofolic acid (5-CHO-(6R,S)-THF), called folinic acid for short, and isolation of N5-formyl-(6S)-5,6,7,8-tetrahydrofolic acid (also named as 5-CHO-(6,S)-THF), the biochemically active citrovorum factor (=growth factor for Leuconostoc citrovorum).
Folinic acid contains 2 centers of asymmetry. In this connection, because folinic acid is synthesized from folic acid, N-(pteroyl)-L-glutamic acid, the optically active C atom contained in the glutamic acid residue is in the L form, whereas the optically active C atom in position 6, which has been produced by hydrogenation of the double bond in the 5,6-position of the pteroyl radical, is in the racemic, the (6R,S), form. Synthetic folinic acid (=leucovorin) accordingly consists of a 1:1 mixture of two diastereomers.
The naturally occurring, e.g., in the liver, folinic acid is solely in the (6S) form as 5-CHO-(6S)-THF. 5-CHO-(6R,S)-THF (folinic acid) is used in the form of its calcium salt (leucovorin calcium) as a pharmaceutical for the treatment of megaloblastic folic acid deficiency anemia, as an antidote for enhancing the tolerability of folic acid antagonists, specifically of aminopterin, methotrexate and fluorouracil, in cancer therapy (leucovorin rescue) and for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis, as well as for enhancing the tolerability of certain antiparasitics, for example trimethoprim-sulfamethoxazole, in chemotherapy. After administration of 5-CHO-(6R,S)-THF, the (6S) content of this diastereomer mixture is rapidly converted into 5-Me-(6S)-THF, whereas the (6R) content is not metabolized and is slowly excreted with the urine: J.A. Straw et al., Cancer Research 44, 3114-3119 (1984).
F.M. Sirotnak et al., Biochemical Pharmacology 28, 2993-97 (1979) have found that the efficacy at suppressing the inhibition of cell growth of methotrexate in L 1210 cultures of the unnatural diastereomer of folinic acid [5-CHO-(6R)-THF] is a 100th, and of the chemically synthesized diastereomer mixture [5-CHO-(6R,S)-THF] is one half that of the natural diastereomer [5-CHO-(6S)-THF]. C. Temple et al., Cancer Treatment Reports 65, 1117-9 (1981), have produced in vivo confirmation of these results obtained in vitro and have found that the natural diastereomer is more than twice as effective as the mixture of diastereomers [leucovorin] with regard to reducing the toxicity of methotrexate. These authors in fact suggested that the unnatural diastereomer may have an injurious effect.
This is because 5-CHO-(6R)-THF inhibits some enzymes responsible for C.sub.1 transfer and thus inhibits the biochemical action of tetrahydrofolates: R.P. Leary et al., Biochem. Biophys. Res. Commun. 56. 484 (1973); V.F. Scott et al., ibid. 14, 523 (1964); G.K. Smith et al., Biochemistry, 20, 4034 (1981). The use of (6S)-tetrahydrofolates in place of (6R,S)-tetrahydrofolates ought not only to be twice as effective but also to have qualitative therapeutic advantages.
Hence, there is a need to replace the 1:1 mixture of diastereomers which has hitherto been used by the natural active substance [5-CHO-(6S)-THF].
Several attempts have been made to resolve 5-CHO-(6R,S)-THF and to carry out the asymmetric synthesis of 5-CHO-(6S)-THF. D. Cosulich et al., J. Amer. Chem. Soc. 74, 4215-16 (1952), U.S. Pat. No. 2,688,018 (Aug. 31, 1954) have attempted, for example, to bring about the resolution by fractional crystallization of an alkaline earth metal salt, for example, the calcium or strontium salt, of 5-CHO-(6R,S)-THF from aqueous solutions [see also J.C. Fontecilla-Camps et al., J. Amer. Chem. Soc. 101, 6114 (1979)].
However, the desired resolution cannot be achieved under the conditions published by D. Cosulich et al. On crystallization of, for example, the calcium salt of 5-CHO-(6R,S)-THF from water at pH 7-8 it is always the 6R,S form which is recovered, as can be demonstrated quantitatively by means of chromatographic analysis on a chiral HPLC column and on the basis of the optical rotation. It is immaterial in this connection whether crude or pure calcium salt of 5-CHO-(6R,S)-THF is used for the crystallization; the (6R,S) form is always recovered. Nor is it possible to achieve resolution and enrichment of the (6S) form by seeding the supersaturated aqueous solution of an alkaline earth metal salt of 5-CHO-(6R,S)-THF with authentic alkaline earth metal salt of 5-CHO-(6S)-THF.
Resolution of the pair of diastereomers has also been attempted by chromatography: J. Feeney et al., Biochemistry 20, 1837 (1981). In addition, the (6S)-isomers have been prepared by stereospecific reduction of dihydrofolates in the presence of dihydrofolate reductase: L. Rees et al., Tetrahedron 42, 117 (1986).
L. Rees et al., J. Chem. Soc., Chem. Commun. 1987, 470, EP-A2 0,266,042 have described a method for resolving (6R,S)-THF, with the aid of which it was possible to produce small amounts of 5-CHO-(6S)-THF and 5-CHO-(6R)-THF. The method comprises reacting (6R,S)-THF with (-)-menthyl chloroformate to give the diastereomeric 5-(-)-menthyloxycarbonyl-tetrahydrofolic acids, resolving the latter by repeated treatment with n-butanol, heating the resulting diastereomers with a saturated solution of hydrogen bromide in a mixture of formic acid and acetic acid, when 5-formyl-(6S)- and (6R)-THF are formed after hydrolysis, and finally isolating the latter as calcium salts. This method is laborious and difficult and requires highly toxic phosgene for the preparation of the chiral reagent. In addition, the starting material (6R,S)-THF is very unstable. On elimination of the chiral auxiliary group with HBr in AcOH at &gt;50.degree. C., there is partial elimination of glutamic acid, resulting in byproducts which can be separated off only with difficulty. The (6S)-folinic acid produced by a method of this type would be so costly that scarcely any consideration would be given to using it in place of (R,S)-tetrahydrofolates.
Thus, the above-described prior art does not disclose any technically and economically attractive method for obtaining (6S)-tetrahydrofolates.